Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
The vascular system is essential for embryonic development and adult life (see, Flamme, I., et al.; Patan, S.,). Once in place, endothelial cells have to maintain blood vessels for life. The natural turnover of endothelial cells is not homogenous throughout the vasculature but concentrated in areas of shear stress (see, Schwartz, S. M. & Benditt, E. P.; Xu, Q,). Based on animal data, endothelial cells in areas resistant to atherosclerosis have a lifespan of 12 months, whereas cells at lesion-prone sites live only for weeks, and even less as animals age. Endothelial cells thus undergo replication many times in some areas of the arterial wall (see, Xu, Q.). This turnover is increased by atherosclerosis and its risk factors, such as stress and age, resulting in endothelial senescence (see, Minamino, T. & Komuro, I.,).
As the endothelial layer is at continuous risk of defects, repair mechanisms are permanently active via endothelial progenitors. These cells may be resident endothelial progenitor cells already in the vessel intima, which divide to produce additional cells. Indeed, highly proliferative endothelial cells can be identified within blood vessels. Vessel-derived resident endothelial cells such as human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) can be passaged in vitro for >40 population doublings—a fact that stands in contrast to the widely held belief that these are terminally-differentiated, mature endothelial cells. Alternatively, endothelial progenitor cells can come from distant sources. A mathematical model estimated that without distant endothelial progenitor cell homing, at least 4.6% (SD 1.0%) of defects in the endothelium could not be repaired by age 65 years (Op den Buijs, J., et al.). This shows however that resident cells undertake the vast majority of vascular repair. This resident endothelial self-renewal capacity depends on age: in murine models 15% of aortic endothelial cells undergo cell division at birth compared to only 0.6% at 6 months (see, Schwartz, S. M. & Benditt, E. P.). The relative contribution of distant endothelial progenitor cells to the vascular endothelium is thus small, but becomes increasingly apparent with advancing age (Op den Buijs, J., et al.). This sheds light on the need for endothelial progenitor cell therapy to maintain adult vascular homeostasis.
The identification of endothelial progenitor cells stimulated numerous clinical trials using different cell types to promote vascular repair, largely on myocardial infarction and critical limb ischemia. The most frequent sources utilized were early outgrowth endothelial progenitor cells from the peripheral circulation and bone marrow mononuclear cells (MNC). Significant improvement was observed after treatment of critical limb ischemia such as reduced limb loss, increased pain-free walking distance and healing of ischemic leg ulcers (see, Kawamoto, A., et al.; Tateishi-Yuyama, E., et al.). The benefits of endothelial progenitor cell therapy in myocardial infarction effects were modest, with improvement of left ventricular ejection fraction (LVEF) by 2-8% (see, Assmus, B., et al.; Stamm, C., et al.) associated with a reduction in the infarction size. However, no study reported any impact of endothelial progenitor cell therapy on major cardiovascular outcomes such as death, re-infarction or stroke (see, Kumar, A. H. & Caplice, N. M.).
A major limitation in these early clinical studies relate to how endothelial progenitor cells were defined. Flow cytometry with CD34, VEGFR2 (KDR/FLK-1) and/or CD133 is conventionally used to identify the number of circulating endothelial progenitor cells, in addition to more classical endothelial markers such as vascular endothelial (VE)-cadherin or CD31. However, no combination of markers has produced a reliable or discriminatory marker set. A recent controlled trial compared the efficacy of sorted or unsorted bone-marrow cells in patients who had recently suffered a myocardial infarction and found no differences in outcomes as both groups improved left ventricle ejection fraction, in similar modest proportions (3%) (see, Tendera, M., et al.). An alternate approach to isolate endothelial progenitor cells is employing partially differentiated endothelial progenitor cells after short term culture on fibronectin, resulting in spindle shaped cells able to digest acetylated low density lipoprotein and stain for several specific lectins appearing within 3 days. Both methods however result in considerable contamination by hematopoietic cells. Asahara et al. reported that the CD34-enriched population was almost exclusively (97%) hematopoietic as they expressed CD45. CD34, VEGFR2 and CD133, significantly enrich for hematopoietic stem cells and are not specific for endothelial progenitor cells (see, Case, J., et al.). Of interest, monocytes (CD34+CD14+) when used in clinical trials have been implicated in the restenosis of revascularized coronary arteries and contribute to atherosclerotic plaques in vessels (see, Kumar, A. H. & Caplice, N. M.; Bartunek, J., et al.). Thus contaminating cells may negate the benefit of pure endothelial progenitor cell therapy.
Another major issue identified in previous trials was the difficulty in achieving adequate cell numbers for therapy. Most regimens used a single injection, whereas continuous delivery would be expected to be advantageous. Circulating endothelial cells isolated by FACS or magnetic sorting (hematopoietic contaminated) have been estimated at 0 to 10 cells/mL blood (see, Woywodt, A., et al.). However, the majority of these peripherally derived cells has limited expansion capacity (17 fold) and the only cells with high proliferative potential (1000 fold) are bone marrow-derived (see, Lin, Y., et al.). Accordingly, these cells are not currently a reliable source for therapies.
In a related aspect, it has also been determined that the isolation method of the present invention, in addition to enabling the isolation of endothelial progenitor cells, enables the isolation of mesenchymal stem cells, in particular fetal mesenchymal stem cells which are not contaminated with maternal mesenchymal stem cells. In fact, two populations of mesenchymal stem cells can be obtained, one that comprises only fetal mesenchymal stem cells and another that upon culturing results in maternal mesenchymal stem cells. Specifically, in the context of a single isolation protocol, multiple distinct and rare precursor cell populations are able to be effectively isolated by virtue of their separation into distinct fractions during the isolation protocol. The development of this protocol therefore provides, for the first time, a reliable and routine means of isolating multiple distinct precursor cell populations.
There is a need therefore to identify an accessible, reliable and abundant source of endothelial progenitor cells if progenitor cell-based vascular therapy is to become a reality. In work leading up to the present invention, an isolation method has been developed which can efficiently and reliably isolate a more pure endothelial progenitor cell population, than has been available to date, which population does not contain the degree of cellular contamination that has characterized prior art isolation methods. This finding thereby provides, for the first time, means to routinely and reliably isolate a pure endothelial cell population for use either in vitro or in vivo. In a further aspect, it has also been determined that the placenta in fact provides a rich source of endothelial cells and that the specific application of this method to the placenta enables isolation of endothelial progenitor cells of both a level of purity and abundance that has not been previously achievable. Accordingly, the application of the isolation method of the present invention on placental tissue, in particular, provides an abundant source of endothelial progenitor cells that exhibit both self-renewal and differentiation capacity. This finding has therefore now enabled the realistic development of endothelial progenitor cell based therapies.